Diffuse Large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Despite recent progress, a large fraction of these patients still die of their disease. The hallmar of the most aggressive subtype of DLBCL, Activated B-cell (ABC)-like DLBCL, is constitutive activation of the NF-B pathway. In approximately half of these cases, aberrant NF-B activation results from somatic mutations in negative and positive regulators of this pathway. However, in a large proportion of NF-B-addicted DLBCLs the molecular basis for these abnormalities is unknown. MicroRNAs (miRNAs) are non-protein coding genes that regulate the human transcriptome by pairing to the 3'UTR of target genes, inducing RNA cleavage and/or translational inhibition. MiRNAs are frequently disrupted in DLBCL, suggesting that they may play a role in constitutively activating NF-kB signals in these tumors. Concordantly, evidence from other biological systems indicates that there is an extensive interplay between miRNAs and the NF-B pathway. The overarching objective of this proposal is to systematically test the novel hypothesis that genetic and epigenetic disruption of the interactions between miRNAs and members of the NF-B pathway contributes to the constitutive activation of these signals in the fatal subtypes of DLBCL. More specifically, we will: 1. Characterize and functionally validate the direct interaction between the DLBCL-relevant miR-125a/b and miR-18a, miR-19a/b with the tumor suppressor gene, and negative NF-B regulator, TNFAIP3; 2. Identify and characterize the miRNAs that directly target the positive NF-B regulators CARD11, MYD88, CD79B, TRAF2, TRAF5 and TAK1; 3. Define the role of the Lin28b and the let-7 miRNA family in an epigenetically-driven self-sustained loop that could constitutively activate NF-B in DLBCL. To achieve the goals of this research proposal, we will make extensive use of genetically modified DLBCL cell lines, in vitro and in vivo, and primary lymphoma samples. In brief, miRNA expression and function will be modulated with stable expression of their precursor sequence via a retrovirus system (gain-of-function), or with miRNA sponges, constructs that sequester the miRNAs away from endogenous binding sites resulting in a significant functional knockdown (loss-of-function). Furthermore, stable ectopic expression and shRNA-mediated knockdown of LIN28B will be used in complementary fashion to define the presence of an epigenetically-driven positive feedback loop that maintains NF-B constitutive active in DLBCL. Throughout this study, the models that yield relevant result in vitro will be validated in a xenograft model o human lymphoma with non-invasive imaging capability. Finally, the relevance of these results will be confirmed in the most critical setting, primary tumors, via measurement of miRNAs, target genes expression and characterization of NF-B activity. The identification of miRNAs that when disrupted activate NF-B signals will clarify the function of multiple oncogenic and tumor suppressive miRNAs and advance our understanding of normal and malignant lymphocyte biology. These findings will elucidate the molecular basis for a substantial fraction of DLBCLs that are addicted to NF-kB signals, an important knowledge gap to be addressed as it relates to the fatal subsets of DLBCLs. Further, since high NF-kB activity is not exclusive of ABC-DLBCL, but contributes to the pathogenesis of multiple mature B-cell malignancies as well as epithelial cancer, our data is likely to have broad relevance and eventually benefit a large patient population. Finally, as the potential of inhibiting and restoring miRNA levels with therapeutic intent is becoming a reality, our studies may provide a blueprint for testing this concept in cancer.